WO2000068140A1 - 623rr - Google Patents

623rr Download PDF

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Publication number
WO2000068140A1
WO2000068140A1 PCT/US2000/012060 US0012060W WO0068140A1 WO 2000068140 A1 WO2000068140 A1 WO 2000068140A1 US 0012060 W US0012060 W US 0012060W WO 0068140 A1 WO0068140 A1 WO 0068140A1
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WIPO (PCT)
Prior art keywords
polypeptide
seq
polynucleotide
sequence
compnsmg
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PCT/US2000/012060
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English (en)
Inventor
Weonhye Bae
Stephanie Van Horn
Richard L. Warren
Sanjoy Biswas
John P. Throup
Martin K. R. Burnham
Original Assignee
Smithkline Beecham Corporation
Smithkline Beecham Plc
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Publication of WO2000068140A1 publication Critical patent/WO2000068140A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to newly identified polynucleotides and polypeptides. and their production and uses, as well as their variants, agonists and antagonists, and their uses
  • the invention relates to polynucleotides and polypeptides of the Two Component Signal Transducuon Response Regulator family, as well as their variants, herein referred to as "623RR.”
  • 623RR polynucleotide(s)," and “623RR polypeptide(s) as the case may be
  • Staphylococci make up a medically important genera of microbes They are known to produce two types of disease, invasive and toxigemc Invasive infections are characterized generally by abscess formation effecting both skin surfaces and deep tissues S aureus is the second leading cause of bacteremia in cancer patients Osteomyelitis, septic arthritis, septic thrombophlebitis and acute bacterial endocarditis are also relatively common There are at least three clinical conditions resulting from the toxigemc properties of Staphylococci The manifestation of these diseases result from the actions of exotoxms as opposed to tissue invasion and bacteremia These conditions include Staphylococcal food poisoning, scalded skin syndrome and toxic shock syndrome
  • Staphylococcus aureus infections has risen dramatically in the past few decades This has been attributed to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems It is no longer uncommon to isolate Staphylococcus aureus strains that are resistant to some or all of the standard antibiotics This phenomenon has created an unmet medical need and demand for new anti-microbial agents, vaccines, drug screening methods, and diagnostic tests for this organism
  • polynucleotides and polypeptides such as the 623 RR embodiments of the invention, that have a present benefit of, among other things, being useful to screen compounds for antimicrobial activity.
  • Such factors are also useful to determine their role in pathogenesis of infection, dysfunction and disease.
  • identification and characterization of such factors and their antagonists and agonists to find ways to prevent, ameliorate or correct such infection, dysfunction and disease.
  • the present invention relates to 623RR, in particular 623RR polypeptides and 623RR polynucleotides. recombinant materials and methods for their production.
  • the invention relates to methods for using such polypeptides and polynucleotides, including treatment of microbial diseases, amongst others.
  • the invention relates to methods for identifying agonists and antagonists using the materials provided by the invention, and for treating microbial infections and conditions associated with such infections with the identified agonist or antagonist compounds.
  • the invention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting 623RR expression or activity.
  • the invention relates to 623 RR polypeptides and polynucleotides as described in greater detail below.
  • the invention relates to polypeptides and polynucleotides of a 623R-R o ⁇ Staphylococcus aureus, that is related by amino acid sequence homology to YesN polypeptide.
  • the invention relates especially to 623RR having a nucleotide and amino acid sequences set out in Table 1 as SEQ ID NO:l and SEQ ID NO:2 respectively.
  • sequences recited in the Sequence Listing below as "DNA” represent an exemplification of the invention, since those of ordinary skill will recognize that such sequences can be usefully employed in polynucleotides in general, including ribopolynucleotides.
  • NCIMB National Collections of Indust ⁇ al and Ma ⁇ ne Bacteria Ltd. 23 St Machar Dnve Aberdeen AB2 1RY, Scotland on 11 September 1995 and assigned NCIMB Deposit No 40771, and referred to as Staphylococcus aureus WCUH29 on deposit
  • the Staphylococcus aureus strain deposit is referred to herein as "the deposited strain” or as "the DNA of the deposited strain "
  • the deposited strain comprises a full length 623 RR gene
  • the sequence of the polynucleotides comp ⁇ sed in the deposited strain, as well as the amino acid sequence of any polypeptide encoded thereby, are controlling in the event of any conflict with anv desc ⁇ pticn of sequences herein
  • the deposit of the deposited strain has been made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure
  • the deposited strain will be irrevocably and without restriction or condition released to the public upon the issuance of a patent
  • the deposited strain is provided merelv as convenience to those of skill in the art and is not an admission that a deposit is required for enablemem. such as that required under 35 U S C ⁇ 112.
  • a license may be required to make, use or sell the deposited strain, and compounds de ⁇ ved therefrom, and no such license is hereby granted
  • an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Staphylococcus aureus WCUH 29 strain, which polypeptide is comp ⁇ sed in the deposited strain
  • 623 RR polynucleotide sequences in the deposited strain such as DNA and RNA, and amino acid sequences encoded thereby
  • 623RR polypeptide of the invention is substantially phylogenetically related to other proteins of the Two Component Signal Transduction Response Regulator family
  • polypeptides o ⁇ Staphylococcus aureus referred to herein as "623 RR” and “623RR polypeptides” as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful va ⁇ ants thereof, and compositions compnsing the same
  • the present invention further provides for an isolated polypeptide that (a) comprises or consists of an ammo acid sequence that has at least 95% identity, most preferably at least 97-99% or exact identity, to that of SEQ ID NO 2 over the entire length of SEQ ID NO 2, (b) a polypeptide encoded by an isolated polynucleotide compnsing or consistmg of a polynucleotide sequence that has at least 95% identity, even more preferably at least 97-99% or exact identit to SEQ ID NO 1 over the entire length of SEQ ID NO 1, (c) a polypeptide encoded by an isolated poh ⁇ ucleot ⁇ de compnsing or consistmg of a polynucleotide sequence encoding a polypeptide that has at least 95% identity, even more preferably at least 97-99% or exact identity, to the amino acid sequence of SEQ ID NO 2. over the entire length of SEQ ED N02
  • polypeptides of the invention include a polypeptide of Table 1 [SEQ ID NO 2] (in particular a mature polypeptide) as well as polypeptides and fragments, particularly those that has a biological activity of 623RR, and also those that have at least 95% identity to a polypeptide of Table 1 [SEQ ID NO 2] and also include portions of such polypeptides with such portion of the polypeptide generally comprising at least 30 amino acids and more preferably at least 50 amino acids
  • the mvention also mcludes a polypeptide consisting of or compnsing a polypeptide of the formula X-(R 1 ) m -(R 2 )-(R 3 ) n -Y wherein, at the ammo te ⁇ nus, X is hydrogen, a metal or any other moiety descnbed herein for modified polypeptides.
  • Y is hydrogen, a metal or any other moiety descnbed herein for modified polypeptides
  • Ri and R3 are any amino acid residue or modified ammo acid residue
  • m is an integer between 1 and 1000 or zero
  • n is an integer between 1 and 1000 or zero
  • R 2 is an amino acid sequence of the mvention, particularly an amino acid sequence selected from Table 1 or modified forms thereof In the formula above, R 2 is onented so that its ammo terminal ammo acid residue is at the left, covalently bound to R j and its carboxy terminal ammo acid residue is at the nght covalently bound to R3 Any stretch of ammo acid residues denoted bv either Ri or R3, where m and/or n is greater than 1.
  • n is an integer between 1 and 50, 100, or 500
  • a polypeptide of the mvention is de ⁇ ved from Staphylococcus aureus, however, it may preferably be obtained from other organisms of the same taxonomic genus
  • a polypeptide of the invention may also be obtained, for example, from organisms of the same taxonomic family or order
  • a fragment is a va ⁇ ant polypeptide having an ammo acid sequence that is entirely the same as part but not all of any ammo acid sequence of any polypeptide of the mvention
  • fragments may be "free-standing,” or comp ⁇ sed within a larger polypeptide of which they form a part or region, most preferably as a single continuous region in a smgle larger polypeptide
  • Preferred fragments include, for example, truncation polypeptides having a portion of an ammo acid sequence of Table 1 [SEQ ID NO 2], or of vanants thereof, such as a continuous senes of residues that mcludes an ammo- and/or carboxyl-terminai ammo acid sequence Degradation forms of the polypeptides of the mvention produced by or m a host cell, particularly a Staphylococcus aureus, are also prefened Further preferred are fragments characterized by structural or functional attnbutes such as fragments that comp ⁇ se alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-formmg regions, coil and co ⁇ -forming regions, hydrophihc regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic mdex regions
  • fragments include an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous ammo acids from the amino acid sequence of SEQ ID NO 2, or an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated or deleted from the amino acid sequence of SEQ ID NO:2.
  • Fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these variants may be employed as intermediates for producing the full-length polypeptides of the invention.
  • Polynucleotides It is an object of the invention to provide polynucleotides that encode 623RR polypeptides, particularly polynucleotides that encode a polypeptide herein designated 623 RR.
  • the polynucleotide comprises a region encoding 623RR polypeptides comprising a sequence set out in Table 1 [SEQ ID NO:l] that includes a full length gene, or a variant thereof.
  • SEQ ID NO:l a sequence set out in Table 1 [SEQ ID NO:l] that includes a full length gene, or a variant thereof. The Applicants believe that this full length gene is essential to the growth and/or survival of an organism that possesses it, such as Staphylococcus aureus.
  • isolated nucleic acid molecules encoding and/or expressing 623 RR polypeptides and polynucleotides, particularly Staphylococcus aureus 623RR polypeptides and polynucleotides, including, for example, unprocessed RNAs, ribozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs.
  • Further embodiments of the invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful polynucleotides and polypeptides, and variants thereof, and compositions comprising the same.
  • Another aspect of the invention relates to isolated polynucleotides, including at least one full length gene, that encodes a 623RR polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID NO:2] and polynucleotides closely related thereto and variants thereof.
  • a 623RR polypeptide from Staphylococcus aureus comprising or consisting of an amino acid sequence of Table 1 [SEQ ID NO: 2], or a variant thereof.
  • a polynucleotide of the invention encoding 623RR polypeptide may be obtained using standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bacteria using Staphylococcus aureus WCUH 29 cells as starting material, followed by obtaining a full length clone.
  • standard cloning and screening methods such as those for cloning and sequencing chromosomal DNA fragments from bacteria using Staphylococcus aureus WCUH 29 cells as starting material, followed by obtaining a full length clone.
  • a polynucleotide sequence of the invention such as a polynucleotide sequence given in Table 1 [SEQ ID NO: l]
  • coli or some other suitable host is probed with a radiolabeled oligonucleotide, preferably a 17-mer or longer, derived from a partial sequence.
  • Clones carrying DNA identical to that of the probe can then be distinguished using stringent hybridization conditions.
  • sequencing the individual clones thus identified by hybridization with sequencing primers designed from the onginal polypeptide or polynucleotide sequence it is then possible to extend the polynucleotide sequence m both directions to determine a full length gene sequence Convemently, such sequencmg is performed, for example, usmg denatured double stranded DNA prepared from a plasmid clone Suitable techniques are descnbed by Maniatis. T .
  • the present mvention provides for an isolated polynucleotide compnsmg or consistmg of (a) a polynucleotide sequence that has at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID NO 1 over the entire length of SEQ ID NO 1, (b) a polynucleotide sequence encoding a polypeptide that has at least 95% identity, even more preferably at least 97-99% or 100% exact, to the ammo acid sequence of SEQ ID NO 2, over the entire length of SEQ ID NO 2
  • a polynucleotide encoding a polypeptide of the present mvention may be obtamed by a process that compnses the steps of screening an appropnate library under stringent hyb ⁇ dization conditions with a labeled or detectable probe consistmg of or compnsmg the sequence of SEQ ED NO 1 or a fragment thereof, and isolating a full-length gene and/or genomic clones compnsmg said polynucleotide sequence
  • the mvention provides a polynucleotide sequence identical over its entire length to a coding sequence (open reading frame) m Table 1 [SEQ ID NO 1] Also provided by the mvention is a coding sequence for a mature polypeptide or a fragment thereof, by itself as well as a coding sequence for a mature polypeptide or a fragment m reading frame with another coding sequence, such as a sequence encoding a leader or secretory sequence, a pre-, or pro- or prepro-protem sequence
  • the polynucleotide of the mvention may also compnse at least one non-coding sequence, including for example, but not limited to at least one non-coding 5' and 3' sequence, such as the transcnbed but non-translated sequences, terrnination signals (such as rho-dependent and rho-rndependent termination signals), nbosome binding sites, Kozak sequences, sequences that stabilize mRNA inarms, and polyadeny
  • a preferred embodiment of the mvention is a polvnucleoude of consisting of or compnsmg nucleotide 757 to the nucleotide immediately upstream of or mcluding nucleotide 1 set forth m SEQ ED NO 1 of Table 1. both of that encode a 623RR polypeptide
  • the mvention also mcludes a polynucleotide consisting of or compnsmg a polynucleotide of the formula
  • R is onented so that its 5' end nucleic acid residue is at the left, X is hydrogen, a metal or a modified nucleotide residue, or together with Y defines a covalent bond, and at the 3' end of the molecule, Y is hydrogen, a metal, or a modified nucleotide residue, or together with X defines the covalent bond, each occurrence of R and R3 is mdependently any nucleic acid residue or modified nucleic acid residue, m is an mteger between 1 and 3000 or zero , n is an mteger between 1 and 3000 or zero, and R 2 is a nucleic acid sequence or modified nucleic acid sequence of the mvention, particularly a nucleic acid sequence selected from Table 1 or a modified nucleic acid sequence thereof In the polynucleotide formula above, R is onented so that its 5' end nucleic acid residue is at the left,
  • m is an mteger between 1 and 50, 100 or 500
  • n is an mteger between 1 and 50, 100, or 500
  • a polynucleotide of the mvention is denved from Staphylococcus aureus, however, it may preferably be obtamed from other organisms of the same taxonomic genus
  • a polynucleotide of the mvention may also be obtamed, for example, from organisms of the same taxonomic family or order
  • polynucleotide encoding a polypeptide encompasses polynucleotides that mclude a sequence encoding a polypeptide of the mvention, particularly a bacte ⁇ al polypeptide and more particularly a polypeptide of the Staphylococcus aureus 623 RR having an ammo acid sequence set out in Table 1 [SEQ ID NO 2]
  • the term also encompasses polynucleotides that mclude a single continuous region or discontinuous regions encoding the polypeptide (for example, polynucleotides interrupted by integrated phage, an integrated insertion sequence, an integrated vector sequence, an integrated transposon sequence, or due to RNA editing or genomic DNA reorganization) together with additional regions, that also may compnse coding and or non-coding sequences
  • the mvention further relates to vanants of the polynucleotides descnbed herein that encode va ⁇ ants of a polypeptide having a deduced ammo acid sequence of Table 1 [SEQ ED NO 2] Fragments of polynucleotides of the mvention may be used, for example, to synthesize full-length polynucleotides of the mvention
  • prefened embodiments are polynucleotides encoding 623RR vanants, that have the ammo acid sequence of 623RR polypeptide of Table 1 [SEQ ED NO 2] m which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no ammo acid residues are substituted, modified, deleted and or added, m any combination Especially prefened among these are silent substitutions, additions and deletions, that do not alter the properties and activities of 623RR polypeptide
  • Prefened isolated polynucleotide embodiments also mclude polynucleotide fragments, such as a polynucleotide comprising a nuclic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids from the polynucleotide sequence of SEQ ID NO: l, or an polynucleotide comprising a nucleic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids truncated or deleted from the 5' and/or 3' end of the polynucleotide sequence of SEQ ID NO:l.
  • polynucleotide fragments such as a polynucleotide comprising a nuclic acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous nucleic acids from the polynucleotide sequence of SEQ ID NO: l, or an polynucleotide comprising a nucleic acid sequence having at least 15, 20, 30, 40, 50 or 100 contig
  • prefened embodiments of the mvention are polynucleotides that are at least 95% or 97% identical over their entire length to a polynucleotide encoding 623 RR polypeptide having an a mo acid sequence set out m Table 1 [SEQ ID NO 2], and polynucleotides that are complementary to such polynucleotides.
  • Most highly prefened are polynucleotides that compnse a region that is at least 95% are especially prefened.
  • those with at least 97% are highly prefened among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly prefened, with at least 99% being the more prefened
  • Prefened embodiments are polynucleotides encoding polypeptides that retain substantially the same biological function or activity as a mature polypeptide encoded by a DNA of Table 1 [SEQ ID NO 1]
  • polynucleotides that hyb ⁇ dize. particularly under stringent conditions to 623RR polynucleotide sequences, such as those polynucleotides m Table 1
  • the mvention further relates to polynucleotides that hybndize to the polynucleotide sequences provided herein
  • the mvention especially relates to polynucleotides that hyb ⁇ dize under strmgent conditions to the polynucleotides descnbed herein
  • the terms "stringent conditions” and “stringent hybndization conditions” mean hyb ⁇ dization occurring only if there is at least 95% and preferably at least 97% identity between the sequences
  • strmgent hybndization conditions is overnight incubation at 42°C m a solution compnsmg 50% formamide, 5x SSC (150mM NaCl, 15
  • the mvention also provides a polynucleotide consistmg of or compnsmg a polynucleotide sequence obtamed by screening an approp ⁇ ate library compnsmg a complete gene for a polynucleotide sequence set forth m SEQ ID NO 1 under strmgent hybndization conditions with a probe havmg the sequence of said polynucleotide sequence set forth m SEQ ID NO 1 or a fragment thereof, and isolating said polynucleotide sequence Fragments useful for obtaining such a polynucleotide mclude. for example, probes and primers fully descnbed elsewhere herem
  • the polynucleotides of the mvention may be used as a hyb ⁇ dization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding 623 RR and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity, to a 623RR gene
  • Such probes generally will compnse at least 15 nucleotide residues or base parrs
  • such probes will have at least 30 nucleotide residues or base pairs and may have at least 50 nucleotide residues or base pairs
  • Particularly prefened probes will have at least 20 nucleotide residues or base pairs and will have lee than 30 nucleotide residues or base pairs
  • a coding region of a 623RR gene may be isolated by screening using a DNA sequence provided m Table 1 [SEQ ID NO 1] to synthesize an oligonucleotide probe
  • a labeled oligonucleoude having a sequence complementary to that of a gene of the mvention is then used to screen a library of cDNN genomic DNA or mRNA to determine which members of the library the probe hybndizes to
  • polynucleotides and polypeptides of the mvention may be employed, for example, as research reagents and mate ⁇ als for discovery of treatments of and diagnostics for diseases, particularly human diseases, as further discussed herem relating to polynucleotide assays
  • polynucleotides of the mvention that are o gonucleotides denved from a sequence of Table 1 [SEQ ID NOS 1 or 2] may be used m the processes herem as descnbed, but preferably for PCR, to determine whether or not the polynucleotides identified herem m whole or m part are transcnbed m bacte ⁇ a in infected tissue It is recognized that such sequences will also have utility m diagnosis of the stage of infection and type of infection the pathogen has attained
  • the mvention also provides polynucleotides that encode a polypeptide that is a mature protem plus additional ammo or carboxyl-terminai ammo acids, or ammo acids mtenor to a mature polypeptide (when a mature form has more than one polypeptide chain, for instance)
  • Such sequences may play a role in processmg of a protem from precursor to a mature form, may allow protem transport, may lengthen or shorten protem half-life or may facilitate rnanipulation of a protem for assay or production, among other things
  • the additional ammo acids may be processed away from a mature protem by cellular enzymes
  • a precursor protem havmg a mature form of the polypeptide fused to one or more prosequences ma ⁇ ' be an inactive form of the polypeptide
  • inactive precursors generalh are activated
  • the mvention also relates to vectors that compnse a polynucleotide or polynucleotides of the mvention, host cells that are genetically engmeered with vectors of the mvention and the production of polypeptides of the mvention by recombinant techniques
  • Cell-free translation systems can also be employed to produce such proteins using RNAs denved from the DNA constructs of the mvention
  • Recombinant polypeptides of the present mvention may be prepared by processes well known m those skilled m the art from genetically engmeered host cells compnsmg expression systems
  • the present mvention relates to expression systems that compnse a polynucleotide or polynucleotides of the present mvention, to host cells that are genetically engmeered with such expression systems, and to the production of polypeptides of the mvention by recombinant techniques For recombinant production of the polypeptides of the mvention
  • approp ⁇ ate hosts include bacte ⁇ al cells, such as cells of streptococci, staphylococci, enterococci E coll, streptor ces, cyanobactena, Bacillus subtilis, and Staphylococcus aureus, fungal cells, such as cells of a yeast, Kluveromyces, Saccharomyces, a basidiomycete, Candida albicans and Aspergillus, insect cells such as cells of Drosophila S2 and Spodoptera Sf9, animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanoma cells, and plant cells, such as cells of a gymnosperm or angjosperm
  • vectors mclude, among others, chromosomal-, episomal- and vrrus-denved vectors, for example, vectors
  • the expression system constructs may compnse control regions that regulate as well as engender expression Generally, any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide m a host may be used for expression m this regard
  • the appropnate DNA sequence may be inserted mto the expression system by any of a vanety of well-known and routine techmques, such as, for example, those set forth m Sambrook et al , MOLECU
  • appropnate secretion signals may be incorporated mto the expressed polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals
  • Polypeptides of the mvention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectm chromatography Most preferably, high performance hqmd chromatography is employed for purification Well known techmques for refoldmg protem may be employed to regenerate active conformation when the polypeptide is denatured du ⁇ ng isolation and or purification
  • This mvention is also related to the use of 623 RR polynucleotides and polypeptides of the mvention for use as diagnostic reagents Detection of 623 RR polynucleotides and/or polypeptides m a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of disease, staging of disease or response of an infectious organism to drugs Eukarvotes. particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism compnsmg the 623RR gene or protein, may be detected at the nucleic acid or amino acid level by a variety of well known techniques as well as by methods provided herein.
  • Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putatively infected and/or infected individual's bodily materials.
  • Polynucleotides from any of these sources may be used directly for detection or may be amplified enzymatically by using PCR or any other amplification technique prior to analysis.
  • RNA, particularly mRNA, cDNA and genomic DNA may also be used in the same ways.
  • amplification, characterization of the species and strain of infectious or resident organism present in an individual may be made by an analysis of the genotype of a selected polynucleotide of the organism.
  • Deletions and insertions can be detected by a change in size of the amplified product in comparison to a genotype of a reference sequence selected from a related organism, preferably a different species of the same genus or a different strain of the same species.
  • Point mutations can be identified by hybridizing amplified DNA to labeled 623 RR polynucleotide sequences. Perfectly or significantly matched sequences can be distinguished from imperfectly or more significantly mismatched duplexes by DNase or RNase digestion, for DNA or RNA respectively, or by detecting differences in melting temperatures or renaturation kinetics.
  • Polynucleotide sequence differences may also be detected by alterations in the electrophoretic mobility of polynucleotide fragments in gels as compared to a reference sequence. This may be carried out with or without denaturing agents. Polynucleotide differences may also be detected by direct DNA or RNA sequencing. See, for example, Myers et al, Science, 230: 1242 (1985). Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase, VI and S 1 protection assay or a chemical cleavage method. See, for example, Cotton et al, Proc. Natl. Acad. Sci., USA, 85: 4397-4401 (1985).
  • an array of oligonucleotides probes comprising 623RR nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of, for example, genetic mutations, serotype, taxonomic classification or identification.
  • Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see, for example, Chee et al, Science, 274: 610 (1996)).
  • the present invention relates to a diagnostic kit that comprises: (a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO:l, or a fragment thereof ; (b) a nucleotide sequence complementary to that of (a); (c) a polypeptide of the present invention, preferably the polypeptide of SEQ ID NO:2 or a fragment thereof; or (d) an antibody to a polypeptide of the present invention, preferably to the polypeptide of SEQ ID NO:2. It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component.
  • Such a kit will be of use in diagnosing a disease or susceptibility to a Disease, among others.
  • This mvention also relates to the use of polynucleotides of the present mvention as diagnostic reagents Detection of a mutated form of a polynucleotide of the mvention, preferable, SEQ ED NO 1, that is associated w th a disease or pathogenicity will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, a prognosis of a course of disease, a determination of a stage of disease, or a susceptibility to a disease, that results from under-expression, over-expression or altered expression of the polynucleotide
  • Organisms, particularly infectious organisms, carrying mutations m such polynucleotide may be detected at the polynucleotide level by a vanety of techmques. such as those descnbed elsewhere herem
  • a polynucleotide and or polypeptide sequence between organisms possessmg a first phenotype and organisms possessmg a different, second different phenotype can also be determined If a mutation is observed m some or all organisms possessmg the first phenotype but not m any organisms possessmg the second phenotype. then the mutation is likely to be the causative agent of the first phenotype
  • a polynucleotide and/or polypeptide of the mvention may also be detected at the polynucleotide or polypeptide level by a vanety of techmques, to allow for serotyping, for example
  • RT-PCR can be used to detect mutations m the RNA It is particularly prefened to use RT-PCR m conjunction with automated detection systems, such as, for example, GeneScan RNA, cDNA or genomic DNA may also be used for the same purpose, PCR
  • PCR primers complementary' to a polynucleotide encoding 623 RR polypeptide can be used to identify and analyze mutations
  • the mvention further provides these pnmers with 1, 2, 3 or 4 nucleotides removed from the 5' and or the 3' end These pnmers may be used for, among other thmgs, amphfymg 623RR DNA and/or
  • the mvention further provides a process for diagnosing, disease, preferably bactenal infections, more preferably infections caused by Staphylococcus aureus, compnsing determining from a sample denved from an mdividual, such as a bodily matenal.
  • Increased or decreased expression of a 623 RR polynucleotide can be measured usmg any on of the methods well known m the art for the quantitation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting, spectrometry and other hybndization methods
  • a diagnostic assay m accordance with the mvention for detectmg over-expression of 623 RR polypeptide compared to normal control tissue samples may be used to detect the presence of an infection, for example Assay techmques that can be used to determine levels of a 623RR polypeptide.
  • a sample denved from a host, such as a bodily matenal. are w ell-known to those of skill m the art
  • Such assay methods mclude radioimmunoassays, competitive-binding assays, Western Blot analysis, antibody sandwich assays, antibody detection and ELISA assays
  • Polypeptides and polynucleotides of the mvention may also be used to assess the binding of small molecule substrates and hgands in, for example, cells, cell-free preparations, chemical hbranes, and natural product mixtures These substrates and hgands may be natural substrates and hgands or may be structural or functional mimetics See, e g , Cohgan etal , Current Protocols in Immunology 1(2) Chapter 5 (1991) Polypeptides and polynucleotides of the present mvention are responsible for many biological functions, mcluding many disease states, m particular the Diseases herem mentioned It is therefore desirable to devise screening methods to identify compounds that agonize (e g , stimulate) or that antagonize (e g .inhibit) the function of the polypeptide or polynucleotide Accordingly, m a further aspect, the present mvention provides for a method of screening compounds to identify those that agonize or that antagonize the
  • agomsts or antagonists may be employed for therapeutic and prophylactic purposes for such Diseases as herem mentioned
  • Compounds may be identified from a vanety of sources, for example, cells, cell-free preparations, chemical hbranes, and natural product mixtures
  • agomsts and antagomsts so-identified may be natural or modified substrates, hgands, receptors, enzymes, etc , as the case may be, of 623RR polypeptides and polynucleotides, or may be structural or functional mimetics thereof (see Cohgan et al , Current Protocols in Immunology 1 (2) Chapter 5 (1991))
  • the screening methods may simply measure the binding of a candidate compound to the polypeptide or polynucleotide, or to cells or membranes bearing the polypeptide or polynucleotide, or a fusion protem of the polypeptide by means of a label directly or mdirectly associated with the candidate compound Alternatively, the screening method may mvolve competition with a labeled competitor Further, these screening methods may test whether the candidate compound results m a signal generated by activation or inhibition of the polypeptide or polynucleotide, usmg detection systems appropnate to the cells compnsmg the polypeptide or polynucleotide Inhibitors of activation are generally assayed m the presence of a known agomst and the effect on activation by the agomst by the presence of the candidate compound is observed Constitutively active polypeptide and/or constitutively expressed polypeptides and polynucleotides may be employed m screemng methods for mverse agonists, m
  • polypeptides and antibodies that bmd to and/or mteract with a polypeptide of the present mvention may also be used to configure screemng methods for detectmg the effect of added compounds on the production of mRNA and/or polypeptide m cells
  • an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide usmg monoclonal and polyclonal antibodies by standard methods known m the art This can be used to discover agents that may inhibit or enhance the production of polypeptide (also called antagonist or agomst, respectively) from suitably manipulated cells or tissues
  • the mvention also provides a method of screemng compounds to identify those that enhance (agomst) or block (antagonist) the action of 623RR polypeptides or polynucleotides, particularly those compounds that are bacte ⁇ static and/or bactencidal
  • the method of screemng may mvolve high-throughput techmques
  • a synthetic reaction mix to screen for agonists or antagonists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, compnsing 623RR polypeptide and a labeled substrate or hgand of such polypeptide is incubated m the absence or the presence of a candidate molecule that may be a 623RR agomst or antagonist
  • the ability of the candidate molecule to agonize or antagonize the 623RR polypeptide is reflected in decreased binding of the labeled hgand or decreased production of product from such substrate Molecules
  • Polypeptides of the mvention may be used to identify membrane bound or soluble receptors, if any, for such polypeptide, through standard receptor bmdmg techmques known m the art These techmques mclude, but are not limited to, hgand bmdmg and crosslinking assavs m which the polypeptide is labeled with a radioactive isotope (for instance. ⁇ 1), chemically modified (for instance, biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (e.g., cells, cell membranes, cell supernatants, tissue extracts, bodily materials).
  • a source of the putative receptor e.g., cells, cell membranes, cell supernatants, tissue extracts, bodily materials.
  • biophysical techniques such as surface plasmon resonance and spectroscopy. These screening methods may also be used to identify agomsts and antagonists of the polypeptide that compete with the binding of the polypeptide to its receptor(s), if any. Standard methods for conducting such assays are well understood in the art.
  • the fluorescence polarization value for a fluorescently-tagged molecule depends on the rotational conelation time or tumbling rate. Protein complexes, such as formed by 623RR polypeptide associating with another 623 RR polypeptide or other polypeptide. labeled to comprise a fluorescently-labeled molecule will have higher polarization values than a fluorescently labeled monomeric protein. It is prefened that this method be used to characterize small molecules that disrupt polypeptide complexes.
  • Fluorescence energy transfer may also be used characterize small molecules that interfere with the formation of 623RR polypeptide dimers, trimers, tetramers or higher order structures, or structures formed by 623RR polypeptide bound to another polypeptide.
  • 623RR polypeptide can be labeled with both a donor and acceptor fluorophore. Upon mixing of the two labeled species and excitation of the donor fluorophore, fluorescence energy transfer can be detected by observing fluorescence of the acceptor. Compounds that block dimerization will inhibit fluorescence energy transfer.
  • Surface plasmon resonance can be used to momtor the effect of small molecules on 623RR polypeptide self-association as well as an association of 623RR polypeptide and another polypeptide or small molecule.
  • 623RR polypeptide can be coupled to a sensor chip at low site density such that covalently bound molecules will be monomeric.
  • Solution protein can then passed over the 623RR polypeptide -coated surface and specific binding can be detected in real-time by monitoring the change in resonance angle caused by a change in local refractive index.
  • This technique can be used to characterize the effect of small molecules on kinetic rates and equilibrium binding constants for 623RR polypeptide self-association as well as an association of 623RR polypeptide and another polypeptide or small molecule.
  • a scintillation proximity assay may be used to characterize the interaction between an association of 623RR polypeptide with another 623RR polypeptide or a different polypeptide .
  • 623RR polypeptide can be coupled to a scintillation-filled bead. Addition of radio-labeled 623RR polypeptide results in binding where the radioactive source molecule is in close proximity to the scintillation fluid. Thus, signal is emitted upon 623 RR polypeptide binding and compounds that prevent 623RR polypeptide self-association or an association of 623RR polypeptide and another polypeptide or small molecule will diminish signal
  • identifying compounds that bmd to or otherwise mteract with and inhibit or activate an activity or expression of a polypeptide and/or polynucleotide of the mvention compnsmg contacting a polypeptide and/or polynucleotide of the mvention with a compound to be screened under conditions to permit bmdmg to or other mteraction between the compound and the polypeptide and/or polynucleotide to assess the bmdmg to or other mteraction with the compound, such bmdmg or mteraction preferably being associated with a second component capable of providing a detectable signal m response to the bmdmg or mteraction of the polypeptide and/or polynucleotide with the compound, and determining whether the compound bmds to or otherwise interacts with and activates or inhibits an activity or expression of the polypeptide and/or polynucleotide
  • polypeptide and/or polynucleotide of the present mvention may also be used m a method for the structure-based design of an agomst or antagonist of the polypeptide and/or polynucleotide.
  • the present inversion provides methods of treatmg abnormal conditions such as, for instance, a Disease, related to either an excess of. an under-expression of, an elevated activity of, or a decreased activity of 623RR polypeptide and'or polynucleotide
  • a Disease related to either an excess of. an under-expression of, an elevated activity of, or a decreased activity of 623RR polypeptide and'or polynucleotide
  • an inhibitor compound (antagonist) as herem descnbed optionally m combination with a pharmaceutically acceptable earner
  • m an amount effective to inhibit the function and/or expression of the polypeptide and/or polynucleotide such as, for example, by blockmg the bmdmg of hgands, substrates, receptors, enzymes, etc , or by inhibiting a second signal, and thereby alleviating the abnormal condition
  • soluble forms of the polypeptides still capable of bmd
  • expression of the gene encodmg endogenous 623RR polypeptide can be inhibited usmg expression blockmg techmques
  • This blockmg may be targeted against any step m gene expression, but is preferably targeted agamst transcnption and/or translation
  • An examples of a known technique of this sort mvolve the use of antisense sequences, either internally generated or separately administered (see, for example, O'Connor, J Neurochem (1991) 56 560 m
  • Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)
  • oligonucleotides that form tnple helices with the gene can be supplied (see, for example, Lee et al , Nucleic Acids Res (1979) 6 3073, Cooney et al , Science (1988) 241 456, Dervan et al , Science (1991) 251 1360)
  • These ohgomers can be administered per se or the relevant o gomers can be expressed in vivo
  • Each of the polynucleotide sequences provided herem may be used m the discovery and development of antibacte ⁇ al compounds
  • the encoded protein upon expression, can be used as a target for the screemng of antibacte ⁇ al drugs
  • the polynucleotide sequences encodmg the ammo terminal regions of the encoded protem or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of mterest
  • the mvention also provides the use of the polypeptide, polynucleotide, agonist or antagonist of the mvention to interfere with the initial physical mteraction between a pathogen or pathogens and a eukaryotic, preferably mammalian, host responsible for sequelae of infection
  • the molecules of the mvention may be used m the prevention of adhesion of bacteria, m particular gram positive and/or gram negative bactena.
  • eukaryotic, preferably mammalian, extracellular mat ⁇ x protems on in-dwelling devices or to extracellular matrix proteins m wounds to block bactenal adhesion between eukaryotic, preferably mammalian, extracellular matrix protems and bacterial 623RR protems that mediate tissue damage and/or. to block the normal progression of pathogenesis m infections initiated other than bv the implantation of in-dwelling devices or by other surgical techmques
  • RR agomsts and antagomsts preferably bacte ⁇ static or bactencidal agonists and antagomsts
  • the antagomsts and agomsts of the mvention may be employed, for instance, to prevent, inhibit and/or treat diseases
  • Hehcobacter pylori herem "H pylori" bactena infect the stomachs of over one-third of the world's population causmg stomach cancer, ulcers, and gastntis (International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Hehcobacter Pylori (International Agency for Research on Cancer, Lyon, France, http //www uicc ch/ecp/ecp2904 htm)
  • the International Agency for Research on Cancer recently recognized a cause-and-effect relationship between H pylori and gastnc adenocarcinoma, classifying the bacte ⁇ um as a Group I (definite) carcmogen
  • Prefened antimicrobial compounds of the mvention agomsts and antagomsts of 623RR polypeptides and/or polynucleotides found usmg screens provided by the mvention, or known m the art, particularly narrow-spectrum antibiotics
  • Bodily matenal(s) means any matenal denved from an mdividual or from an organism infecting, infesting or inhabiting an mdividual, mcludmg but not limited to, cells, tissues and waste, such as, bone, blood, serum, cerebrospinal fluid, semen, saliva, muscle, cartilage, organ tissue, skin, urine, stool or autopsy mate ⁇ als
  • D ⁇ sease(s) means any disease caused by or related to infection by a bactena, mcludmg , for example, disease, such as. infections of the upper respiratory tract (e g , otitis media, bactenal tracheitis, acute epiglottitis, thyroiditis), lower respiratory (e g , empyema. lung abscess), cardiac (e g , infective endocarditis), gastrointestinal (e g , secretory dianhoea, splenic absces.
  • infections of the upper respiratory tract e g , otitis media, bactenal tracheitis, acute epiglottitis, thyroiditis
  • lower respiratory e g , empyema. lung abscess
  • cardiac e g , infective endocarditis
  • gastrointestinal e g , secretory dianhoea, splenic absces.
  • CNS e g , cerebral abscess
  • eye e g , blepha ⁇ tis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital celluhtis, darcryocystitis
  • kidney and urinary tract e g , epi ⁇ dvrnitis. lntrarenal and pe ⁇ neph ⁇ c absces, toxrc shock syndrome
  • sl ⁇ n e g , impetigo, folhcuhtis, cutaneous abscesses, celluhtis. wound infection, bactenal myositis
  • bone andjomt e g , septic arth ⁇ tis, osteomvehtis
  • “Host cell(s)” is a cell that has been introduced (e g , transformed or transfected) or is capable of mtroduction (e g , transformation or transfection) b ⁇ an exogenous polynucleotide sequence
  • Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be as detenruned by companng the sequences
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be. as determined by the match between strings of such sequences
  • Identity can be readily calculated by known methods, mcludmg but not limited to those descnbed m (Computational Molecular Biology, Lesk, A M .
  • Parameters for polypeptide sequence companson mclude the following Algonthm Needleman and Wunsch, J Mol Biol 48 443-453 (1970) Companson mat ⁇ x BLOSSUM62 from Hentikoff and Hentikoff, Proc Natl Acad Sci USA 89 10915-10919 (1992) Gap Penalty 12 Gap Length Penalty 4 A program useful with these parameters is pubhch available as the "gap" program from Genetics Computer Group, Madison WI The aforementioned parameters are the default parameters for peptide compansons (along with no penalty for end gaps)
  • Polynucleotide embodiments further mclude an isolated polynucleotide compnsmg a polynucleotide sequence havmg at least a 95, 97 or 100% identity to the reference sequence of SEQ ID NO 1, wherem said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO 1 or may mclude up to a certain mteger number of nucleotide alterations as compared to the reference sequence, wherem said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, mcludmg transition and transversion, or insertion, and wherem said alterations may occur at the 5 ' or 3' terminal positions of the reference nucleotide sequence or anywhere between those termmal positions, mterspersed either mdividually among the nucleotides m the reference sequence or in one or more contiguous groups within the reference sequence, and wherem said number of nucleotide alterations is determined by multiplying the total number of nu
  • n n is the number of nucleotide alterations
  • x n is the total number of nucleotides in SEQ ID NO 1
  • y is 0 95 for 95%, 0 97 for 97% or 1 00 for 100%
  • • is the symbol for the multiplication operator
  • any non-mteger product of x n and y is rounded down to the nearest mteger pnor to subtractmg it from x n
  • Alterations of a polynucleotide sequence encodmg the polypeptide of SEQ ID NO 2 may create nonsense, missense or frameshift mutations m this codmg sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations
  • Polypeptide embodiments further mclude an isolated polypeptide compnsmg a polypeptide havmg at least a 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2, wherem said polypeptide sequence may be
  • n a is the number of ammo acid alterations.
  • x a is the total number of ammo acids m SEQ ID NO 2
  • y is 0 95 for 95%, 0 97 for 97% or 1 00 for 100%
  • • is the symbol for the multiplication operator, and wherem any non-mteger product of x a and y is rounded down to the nearest mteger pnor to subtracting it from x a
  • “Ind ⁇ v ⁇ dual(s)" means a multicellular eukaryote.
  • mcludmg but not limited to a metazoan, a mammal, an ovid, a bovid, a simian, a p ⁇ mate, and a human
  • Isolated means altered “by the hand of man” from its natural state, i e , if it occurs m nature, it has been changed or removed from its onginal environment, or both
  • a polynucleotide or a polypeptide naturally present in a living organism is not “ isolated,” but the same polynucleotide or polypeptide separated from the coexisting mate ⁇ als of its natural state is “isolated", as the term is employed herem
  • a polynucleotide or polypeptide that is introduced mto an organism by transformation, genetic mampulation or by any other recombinant method is "isolated” even if it is still present m said organism, which organism may beutz or non-living
  • Orgamsm(s) means a (I) prokaryote, mcludmg but not limited to, a member of the genus Streptococcus, Staphylococcus, Bordetella, Corynebactenum, Mycobactenum, Neissena, Haemophilus, Actinomycetes, Streptomycetes, Nocardia Enterobacter, Yersinia Fancisella, Pasturella, Moraxella, Acinetobacter, Erys ⁇ elothnx, Branhamella Actmobacillus, Streptobacillus, Listena, Calymmatobactenum, Brucella, Bacillus, Clostndium, Treponema, Eschenchia, Salmonella, Kleibsiella, Vibno, Proteus, Erwima, Borrelia, Leptospira Spmllum, Campylobacter, Shigella, Legionella Pseudomonas, Aero
  • Streptococcus faecium Streptococcus durans, Neissena gonorrheae, Neissena memngitidis, Staphylococcus aureus, Staphylococcus epidermidis, Corynebactenum d ⁇ thenae, Gardnerella vaginahs.
  • Mycobactenum tuberculosis Mycobactenum bovis, Mycobactenum ulcerans, Mycobactenum leprae, Actinomyctes israelii, Listena monocytogenes, Bordetella pertusis, Boraatella parapertusis, Bordetella bronchiseptica, Eschenchia coh, Shigella dysentenae, Haemophilus influenzae, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonella typhi, Citrobacter freundu, Proteus mirabilis, Proteus vulgans, Yersima pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratia liquefaciens, Vibno cholera, Shigella dysenteni, Shigella flexnen, Pseudomonas aem
  • Polynucleotide(s) generally refers to any polyribonucleotide or polydeoxy ⁇ bonucleotide, that may be unmodified RNA or DNA or modified RNA or DNA.
  • Polynucleoude(s) include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybnd molecules compnsmg DNA and RNA that may be single-stranded or, more typically, double-stranded, or triple-stranded regions, or a mixture of single- and double-stranded regions.
  • polynucleotide refers to tnple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the strands in such regions may be from the same molecule or from different molecules.
  • the regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules.
  • One of the molecules of a triple-hehcal region often is an oligonucleotide.
  • polynucleotide(s) also includes DNAs or RNAs as described above that comprise one or more modified bases.
  • DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotide(s)" as that term is intended herein.
  • DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as t ⁇ tylated bases, to name just two examples are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art.
  • polynucleotide(s) as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA charactenstic of viruses and cells, mcludmg, for example, simple and complex cells "Polynucleotide(s)” also embraces short polynucleotides often refened to as ohgonucleotide(s)
  • Polypeptide(s) refers to any peptide or protem compnsmg two or more ammo acids joined to each other by peptide bonds or modified peptide bonds
  • Polypeptide(s) refers to both short chains, commonly refened to as peptides, ohgopeptides and ohgomers and to longer chains generally refened to as proteins
  • Polypeptides may compnse ammo acids other than the 20 gene encoded ammo acids
  • Polypeptide(s)” mclude those modified either by natural processes, such as processmg and other post-translational modifications, but also by chemical modification techmques Such modifications are well descnbed m basic texts and m more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art It will be appreciated that the same type of modification may be present m the same or varying degree at several sites m a given polypeptide Also, a given polypeptide may compnse many types of modifications Mod
  • mcludmg the peptide backbone, the ammo acid side-chains, and the ammo or carboxyl termini Modifications mclude, for example, acetylation, acylation, ADP-nbosylation, amidation.
  • Recombinant expression system(s) refers to expression systems or portions thereof or polynucleotides of the mvention introduced or transformed mto a host cell or host cell lysate for the production of the polynucleotides and polypeptides of the mvention 'Nanant(s)" as the term is used herem, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties
  • a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes m the nucleotide sequence of the va ⁇ ant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result m ammo acid substitutions, additions, deletions, fusion protems and truncations m the polypeptide encoded by the reference sequence, as discussed
  • a vanant and reference polypeptide may differ in ammo acid sequence by one or more substitutions, additions, deletions in any combination
  • a substituted or inserted ammo acid residue may or may not be one encoded by the genetic code
  • the present mvention also mcludes mclude vanants of each of the polypeptides of the mvention, that is polypeptides that vary from the referents by conservative ammo acid substitutions, whereby a residue is substituted by another with like characte ⁇ stics Typrcal such substitutions are among Ala, Val, Leu and lie, among Ser and Thr, among the acidic residues Asp and Glu, among Asn and Gin, and among the basic residues Lys and Arg, or aromatic residues Phe and Tyr Particularly prefened are va ⁇ ants m which several, 5-10, 1-5, 1-3, 1-2 or 1 ammo acids are substituted, deleted, or added
  • the polynucleotide havmg a DNA sequence given m Table 1 [SEQ ID NO 1] was obtamed from a library of clones of chromosomal DNA of Staphylococcus aureus m E coh
  • Total cellular DNA is mechanically sheared by passage through a needle m order to size- fractionate accordmg to standard procedures
  • DNA fragments of up to l lkbp m size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added Fragments are hgated mto the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E coh infected with the packaged library
  • the library is amplified by standard procedures
  • Total cellular DNA is partially hydrolyzed with a one or a combmation of rest ⁇ ction enzymes appropnate to generate a senes of fragments for cloning mto library vectors (e g , Rsal, Pall, Alul, Bshl235I), and such fragments are size-fractionated accordmg to standard procedures EcoRI linkers are hgated to the DNA and the fragments then hgated mto the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E coll infected with the packaged library
  • the library is amplified by standard procedures

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Abstract

La présente invention concerne des polypeptides 623RR et des polynucléotides codant pour les polypeptides 623RR, ainsi que des procédés de production de tels polypeptides au moyen de techniques de recombinaison. Elle concerne aussi des procédés d'utilisation de polypeptides 623RR dans le criblage de composés antibactériens.
PCT/US2000/012060 1999-05-06 2000-05-03 623rr WO2000068140A1 (fr)

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WO2017032909A1 (fr) 2015-08-26 2017-03-02 Universidad Pública de Navarra Souches mutantes de staphylococcus aureus avec systèmes tcs inactivés multiples

Citations (1)

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Publication number Priority date Publication date Assignee Title
EP0786519A2 (fr) * 1996-01-05 1997-07-30 Human Genome Sciences, Inc. Polynucléotides et séquences de Staphylococcus aureus

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Publication number Priority date Publication date Assignee Title
EP0786519A2 (fr) * 1996-01-05 1997-07-30 Human Genome Sciences, Inc. Polynucléotides et séquences de Staphylococcus aureus

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Title
BARRETT J.F. ET AL.: "Antibacterial agents that inhibit two-component signal transduction systems", PROC. NATL. ACAD. SCI. USA, vol. 95, no. 9, 28 April 1998 (1998-04-28), pages 5317 - 5322, XP002931853 *
CHAN P.F. ET AL.: "Role of SarA in virulence determinant production and environmental signal transduction in staphylococcus aureus", JOURNAL OF BACTERIOLOGY, vol. 180, no. 23, December 1998 (1998-12-01), pages 6232 - 6241, XP002931854 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017032909A1 (fr) 2015-08-26 2017-03-02 Universidad Pública de Navarra Souches mutantes de staphylococcus aureus avec systèmes tcs inactivés multiples

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